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Comparison of nitrogenase and nitrate reductaseactivities in two nitrogen-fixing tree species: black alder Alnus glutinosa and black locust Robinia pseudoacacia G.. Nitrogenase t’BI2ase

Comparison of nitrogenase and nitrate reductase

activities in two nitrogen-fixing tree species:

black alder (Alnus glutinosa)

and black locust (Robinia pseudoacacia)

G Pizelle S Benamar F Boutekrabt G Thiéry

I Laboratoire de Physiologie Végétale et Forestibre, Facult6 c/es Sciences, BP 239, 54506

Vandœuvre-Iès-Nancy Cedex, and

2Physiologie V6g6tale, ENSAIA, 54500 !anda°wre-/e5-/B/ancy France

Introduction

Among the symbiotic nitrogen-fixing trees

of the temperate zone, black alder (Alnus

glutinosa, L Gaertn.) and black locust

(Robinia pseudoacacia L.) can be

re-garded as typical actinorhizal (frankial)

and leguminous (rhizobial) woody species,

respectively Important differences

be-tween both species concern their

biologi-cal, ecological and symbiotic

characteris-tics, and the greater amount of work

recently devoted to A glutinosa, which

likely prevails over R pseudoacacia on

the following grounds: ubiquity in Europe,

better sylvicultural qualities, absence of

spines and suckers, easier collection of

the nitrogen-fixing organs, attraction to the

more recently recognized actinorhizal

symbiosis (Tjepkema et al., 1986)

Never-theless, R pseudoacacia is also a tree of

interest both for its role as symbiont in

nitrogen fixation and for its potential value

as timber and wood fiber source (Moiroud

and Capellano, 1982; Turvey and

Smeth-urst, 1983); hence, our desire to progress

in understanding of its physiology.

In an extension of our work on nitrogen

nutrition of A glutinosa (e.g., Pizelle and

Thiéry, 1986), the present paper examines

the nitrogenase and nitrate reductase

ac-tivities, measured in vivo, in R

pseudoa-cacia and compares them with the data

from A glutino!;a.

Materials and IMethods

Plant material was harvested in the early afternoon from 1 ! 3-20 yr old black alders

natu-rally growing on sandy alluvium and from black locusts planted for about 10 yr on a sandy and stony bank; both sites were located on a

sili-ceous substrate in the Moselle valley near

Nancy

Nitrogenase (t’BI2ase) activity was assayed by the Creduction method on excised actino-rhizal lobes of alder and on excised nodules of

black locust Nitrate reductase (NR) activities

were assayed on 5 mm sections of small roots

(diameter 1 1 mrr!; 100 mg fresh tissue) and on

disks of young fully expanded leaves (diameter:

disks) samples

vacuum infiltrated in 2.5 ml of 0.1 M NaK

phos-phate buffer, pH 7.5, with or without 0.05 M

KN0

After incubation for 1 h at 30°C in the

dark, 1.5 ml of incubation medium were cleared

by addition of 0.3 ml of 1 M Zn acetate and

cen-trifugation In the leaf NR assays, Triton X-100

was added (0.1%, v/v) to the incubation

medium and clearance of Zn acetate was not

necessary The nitrite concentrations of the NR

incubation media and soil nitrate content were

determined as described by Pizelle and Thi6ry

(1986)

Results

N

ase activity

In A glutinosa the enzyme activity started

earlier in spring and disappeared later in

than in R pseudoacacia During

the growing season, N ase activity of the

nodules of R pseudoacacia reached higher values than that of the actinorhizas

of A glutinosa (Fig 1 This difference

might be explained by the following

char-acteristics: most nodules of black locust

were less than 1 yr old with a large

vol-ume of inner tissues invaded by the active

bacteroids, whereas the alder actinorhizas included lobes of various ages (often more

than 1 yr old) with the tissues containing

the active vesicles of Frankia limited to the

subapical cortical region.

NR activity of the small roots

Root NR activity of both species did not

disappear in winter (Fig 2) The enzyme

activity

relevant site showed higher values in A

glutinosa than in R pseudoacacia In

contrast with the alder roots, the roots of

hI

likely nitrate, since the NR activities measured with and

without KN0 in the incubation medium

were similar

.1 30

At glutinosa site, higher

soil nitrate content and of enzyme activity

were measured during the growing

sea-son, but no significant correlation (r= =

0.25; n = 21 ) was found between these

parameters.

NR activity of the leaves

Previous experiments showed that the leaf

NR activity of field-grown A glutinosa was

high after bud opening in the spring, then

decreased during the growing season,

before disappearing at the leaf fall; the

presence of this NR activity was found to

be independent of the supply of nitrate to

the leaves via the xylem sap (Pizelle and

Thi6ry, 1977 ; 1986

From the present data (Table I), it

appears that field-grown R pseudoacacia

also had a notable leaf NR activity; the

values were lower in spring than in

sum-mer, unlike the pattern observed in A

glu-tinosa It is presently unknown whether

the variations of the leaf enzyme activity

were controlled by the nitrate supply under

field conditions However, assays using

young nodulated black locusts grown on

nutrient solution without nitrate showed an

increase of their leaf NR activity from

0.78 ± 0.16 to 3.23 ± 1.15 nmol N02 !mg-!

DW-h- (means of 4 samples ± SE) after 3

days of 4 mM NaNO supply.

Discussion and Conclusion

Though the age of the trees and the

na-ture or nitrate content of the soil differed between the respective sites of each

spe-cies, the present data reveal some char-acteristics of the in vivo N ase and NR

activities in field-grown A glutinosa and

R pseudoacacia.

N ase activity of the actinorhizas of A

glutinosa lasted longer but reached lower values than that of the nodules of R

pseudoacacia during the growing season.

These differences are probably related to the length of the period of active photo-synthesis and to the anatomical structure

of the nitrogen-fixing organs in each spe-cies Since N ase activity is measured per

mg dry weight of actinorhiza or nodule, further data, such as the mass of

symbio-tic organs per tree, would be necessary to

compare the nitrogen-fixing potential of the trees of both species.

The presence of a root NR activity in winter indicates the persistence of the

enzyme and reducing power in the roots

out of the growing season Since nitrate

was found in the soil, even in winter, it

might suffice to maintain a nitrate-indu-cible NR activity in the roots throughout

the year The presence of non-reduced nitrate in the roots of R pseudoacacia, in

spite of a low nitrate content in the soil, indicates a limited root NR activity in this

species glutinosa, negligible

accumulation of nitrate in the roots and

the absence of a close correlation

bet-ween root NR activity and soil nitrate

content suggest that the roots have an NR

capacity able to reduce higher amounts of

nitrate than those available in the soil

The seasonal profile of the leaf NR

ac-tivity of A glutinosa appears to be

inde-pendent of the nitrate supply; it decreases

from the early leaf expansion to the

approach of the leaf fall In R

pseudoa-cacia the leaf NR activity shows a different

profile with values lower in spring and

higher in summer Given the results

ob-tained from young laboratory-grown plants

as well as the presence of a limited

ni-trate-reducing capacity of the roots, it can

be proposed that the leaves of R

pseu-doacacia have an NR activity

commensu-rate with their supply of nitrate

Moiroud A & Capellano A (1982) Le robinier, Robinia pseudoacacia L., une espbce fixatrice d’azote int6ressante ? Ann Sci For 39,

407-418

Pizelle G & Thiéry G (1977) Variations saison-nieres des activites nitrogenase et nitrate reductase chez I’aune glutineux (Alnus

glutino-sa L Gaertn.) Pl 1 ysiol Veg 15, 333-342 Pizelle G & Thièry G (1986) Reduction of

ni-trate in the perennial tissues of aerial parts of Alnus glutinosa Phys;b/ Plant 68, 347-352

Tjepkema J.D., Schwintzer C.R & Benson D.R.

(1986) Physiology of actinorhizal nodules Annu Reu Plant Physiol 37, 209-232

Turvey N.D & Smethurst P.J (1983) Nitrogen

fixing plants in forest plantation In: Biological

Nitrogen Fixation in Forest Ecosystems: Foun-dations and Applications (Gordon J.C & Wheeler C.T., eds.), Martinus Nijhoff, The Hague, pp 233-260